Heterozygous chromosomal aberrations in the autism susceptibility candidate 2 (AUTS2) gene region, some of which remove only intronic regions, have been associated with autism spectrum disorders (ASDs). In addition, nucleotide variants, primarily noncoding, in the first half of AUTS2 are thought to make up the most significant accelerated genomic region differentiating humans from Neanderthals.
Objectives:
The function and regulation of AUTS2 are largely unknown despite the involvement of this gene in ASD and modern human evolution. Here, we set out to characterize the functional role and regulatory landscape of AUTS2. In addition, we are analyzing the mechanisms by which changes in this gene’s regulatory sequences could lead to ASD and modern human evolution.
Methods:
To characterize AUTS2 function, we used morpholinos (MOs) to knock-down auts2 in zebrafish. To decode the regulation of AUTS2, we identified tissue specific enhancers that may regulate this gene. Using comparative genomics and available ChIP-Seq data sets, we scanned regions around AUTS2 that are associated with ASD or human-Neanderthal sequence changes for potential enhancer elements. Enhancer candidates were tested using a transgenic zebrafish assay and a subset of positive enhancers were verified in a similar mouse enhancer assay.
Results:
Two different auts2 MOs led to a smaller head size, a loss of neurons in the midbrain (including the cerebellum), decreased volume of motor neurons in zebrafish embryos and decreased movement in response to touch. These phenotypes were rescued by co-injection of human AUTS2 mRNA. Further analysis of auts2 morphants revealed an increase in cell death in the embryonic brain. As for AUTS2 regulation, 21 of the 38 candidate sequences function as enhancers in zebrafish and had expression patterns that overlapped with auts2. Three of these were also functional brain enhancers in mice and are within a 33kb noncoding ASD-associated deletion.
Conclusions:
Combined, our results show that AUTS2 is an important neurodevelopmental gene. In addition, they provide a regulatory map for central nervous system (CNS) enhancers that could regulate AUTS2, revealing candidate sequences where nucleotide variation could lead to ASD susceptibility and human specific traits.